Intravascularly administrable, magnetically responsive nanosphere or nanoparticle, a process for the production thereof, and the use thereof

ABSTRACT

An intravascularly administrable, magnetically responsive nanosphere or nanoparticle made up of a crystalline carbohydrate matrix, preferably starch, enclosing a magnetic material, is described. The nanosphere or nanoparticle is produced by dissolving a carbohydrate together with a magnetic material to form a solution which is emulsified in a hydrophobic solvent from which the carbohydrate is crystallized. 
     The resulting magnetic nanospheres having an average diameter not exceeding 1500 nm are capable of transporting pharmacologically active substances and can be injected intravenously for subsequent concentration in a part of the body by means of a magnetic field.

The present invention relates to a magnetically responsive andbiologically degradable nanosphere or nanoparticle for intravascularadministration, to a process for the production thereof, and to the usethereof for the transport and concentration of pharmaceuticals.

BACKGROUND

It has long been desired, in the treatment of different diseases, to beable specifically to deliver a pharmaceutical to a particular spot inthe body, in particular in the treatment of tumours because of the gravesystemic secondary effects produced by cytostatics. Various chemicalmethods have been tested in which it was tried to utilize differences incell structure between tumour cells and healthy cells. However, no suchtest has been shown to give unequivocal results, above all because thedifference between the tumour cells and the healthy cells is soinsignificant.

One way of solving this problem is to utilize magnetic carriers ofpharmaceuticals which are injected into the blood-vessel system andtransported by the blood to be stopped at the target by means of amagnetic field.

U.S. Pat. No. 4,335,094 (priority June 2, 1977) discloses microspherescontaining magnetic material incorporated with a polymer which, inaddition, carries biologically active components.

U.S. Pat. No. 4,247,406 contains a detailed description of the use andproduction of magnetic microspheres made up of a magnetic material whichhas been enclosed in a polymer consisting of albumin.

One highly important reason for enclosing the magnetic material in amatrix, and for not using only the magnetic material, is that a matrixmakes it possible to transport greater amounts of pharmalogically activesubstance per microsphere or microparticle, which is a prior conditionof opening up possibilities of providing therapeutical concentrations ofthe pharmacologically active substances at the target site, withoutincreasing the number of microspheres to such an extent that thecapillaries are occluded. The requirements placed on the matrix used insuch contexts are as follows:

1. The matrix should in itself be chemically inert in biologicalsystems.

2. The matrix should be biologically well-characterised.

3. The matrix should be non-toxic and non-immunogenic.

4. The matrix should be dischargeable from the body via normal routes.

5. The matrix preparation should be readily administrable.

6. The matrix preparation should be able to release a biologicallyactive substance, and the release rate of the active substance should bereadily controllable.

7. The matrix should be usable for enclosing and releasing substanceshaving different molecular weights.

In addition to polymers of amino acids, such as albumin, carbohydratesare conceivable. Starch and glycogen are such carbohydrates made up ofglucose units and satisfying the requirements for biocompatibility,primarily because they are the body's own substances, which means thatsecondary effects in the form of hypersensitivity reactions are avoided.The body's own enzyme for degrading starch is alpha-amylase whichspecifically degrades alpha(1-4) bonds.

The use of starch is disclosed by DE-OS No. 25 24 278 which describesthe preparation and utilization of covalent cross-linked microspheres ofstarch having a size so selected that the microspheres will get caughtin the capillaries and thereby can be utilized as a diagnostic means inthe vascular system. To ensure that these microspheres get caught, thesize of the microsphere must be above 10 μm.

To enable a microsphere injected into the vascular system to circulate,the diameter of the microsphere must be below 1 μm. This has been shownby, inter alia, Okamoto et al (Chem. Pharm. Bull. (1975) 23(7),1452-1457) who also have shown that the surface structure must behydrophilic. The magnetic albumin microspheres previously mentioned arehydrophobic and cannot therefore circulate, but must be injectedarterially towards the target site.

The present specification describes a simple and reproducible processfor the production of magnetically responsive crystalline nanospheres ornanoparticles for the concentration of pharmaceuticals.

DESCRIPTION OF THE INVENTION

The present invention provides a magnetically responsive andbiologically degradable nanosphere or nanoparticle for intravascularadministration, which is made up of a matrix in which a magneticmaterial is enclosed. The nanosphere or nanoparticle is characterised inthat its average diameter does not exceed 1500 nm, and preferably isless than 1000 nm, and in that the matrix is a crystalline carbohydrate.

Carbohydrate polymers containing alpha(1-4) bonds are especially usefulbecause they can be degraded by the alpha-amylase in the body. Althoughstarch is preferred, also pullullan, glycogen and dextran may be used.It is also possible to modify the carbohydrate polymer with, forexample, hydroxyethyl, hydroxypropyl, acetyl, propionyl,hydroxypropanoyl, various derivatives of acrylic acid or likesubstituents.

Also carbohydrates which are not polymeric, may be used in the contextof this invention. Examples of such carbohydrates are glucose, maltoseand lactose.

Pharmaceuticals may be adsorbed to the carbohydrates after thenanosphere has been produced. This may be important in such cases wherethe pharmaceutical in question is damaged by the treatment in connectionwith the production of the magnetic nanospheres. If the matrix is acarbohydrate, it is also possible to modify the matrix by covalentlycoupling to the carbohydrate e.g. amino groups or carboxylic acidgroups, thereby to create an adsorption matrix. High molecularsubstances of the type proteins may be enclosed within the matrix forlater release.

The invention also comprises a process for the production of magneticnanospheres having a diameter below 1.5 μm. The process is characterisedin that a carbohydrate is dissolved in a solvent having a highdielectric constant to form a clear solution. To this solution, themagnetic material is added, whereupon the hydrophilic solution isemulsified in a hydrophobic solvent. The resulting emulsion isstabilized by supplying thereto a stabilizing medium, or by transferringthe emulsion to such a medium, whereby the carbohydrate is crystallized,while enclosing the magnetic material, to magnetic nanospheres ornanoparticles having an average diameter which does not exceed 1500 nmand preferably is less than 1000 nm.

The carbohydrate is dissolved in a concentration which varies from onesolvent to another but which normally lies within the range 5-250%(weight volume). Conceivable such solvents are, inter alia, dimethylformamide, ethylene glycol, sulpholane, dimethyl sulphoxide, propylenecarbonate, water and formamide, or mixtures thereof.

The magnetic material may consist of magnetic magnetite particles (Fe₃O₄) having a size of 10-20 nm. One process for the production of suchsmall particles is known, and the particles are commercially availablefrom Ferrofluid Corp., USA. Other useful magnetic materials includeparticles or colloids having a substantial content of the aluminium,nickel, cobalt, copper, silver, manganese, or platinum.

The amount of magnetic particles in the magnetic nanospheres may varywithin wide limits. In actual practice, however, the range is rathernarrow, and preferably use is made of 10-150 parts by weight of magneticparticles per 100 parts by weight of matrix. The mixture of thedissolved carbohydrate and the magnetic material is emulsified in ahydrophobic phase, resulting in the formation of a W/O emulsion. Thehydrophobic phase employed may be a vegetable oil, preferably maize oil,or an organic solvent in which one or more emulsifiers have beendissolved. Among such useful organic solvents are, inter alia, xylene,toluene, ethyl benzene, diethyl benzene, propyl benzene, ethylenechloride and the like, as well as mixtures thereof. The emulsificationsystem which has been found to give the best results, consists of thesolvents xylene/CHCl₃ (4:1) in which the emulsifiers Pluronic F-68® andPluronic L-35® have been dissolved to a concentration of 2.5% (weightvolume) of each emulsifier.

To emulsify the suspension Sonicator or a high-pressure homogenizer isused. The resulting emulsion in which the matrix with the magneticmaterial is suspended in the form of droplets having a maximum size of 2μm, is stabilized by transferring it to a liquid capable ofcrystallizing the carbohydrate, whereby the magnetic material will beenclosed. Examples of such liquids are ethanol, methanol and acetone.The preferred liquid is acetone in which Tween 80® has been dissolved toa concentration of 0.1% (weight volume). The nanospheres are washed withthe said acetone solution before they are dried by rotationalevaporation or freeze drying. They can also be kept for several monthsin the acetone solution.

The pharmacologically active substances can be incorporated with thecarrier, on one hand by enclosure, in which case the pharmacologicallyactive substance is mixed with the solution of carbohydrate and themagnetic material before the solution is emulsified and, on the otherhand, by adsorption, in which case the pharmacologically activesubstance is mixed with the magnetic nanospheres in the aqueous phase.Alternatively, dried magnetic nanospheres may be added to a solution ofpharmacologically active substance, in which case the substance can becoupled covalently to the carbohydrate matrix.

To enable the pharmacologically active substance to exert its effect, itmust be releasable from the carrier material.

When the pharmacologically active substance is enclosed in and/oradsorbed to the crystallized carbohydrate matrix, release is effected bya combination of diffusion and erosion of the matrix, whereas in thecase of a covalent coupling of the active substance, release isaccomplished by degradation of the matrix.

It is also possible to vary the release rate of the pharmacologicallyactive substance by cross-linking the matrix after crystallization. Thetighter the matrix is cross-linked, the longer are the release times.Different types of cross-linking agents can be used, depending uponwhether or not water is present at the cross-linkage. In aqueousenvironment, it is possible to use, inter alia, divinyl sulphone,epibromohydrin or BrCN. In the anhydrous phase, it is possible toactivate with tresyl reagent, followed by cross-linking with a diamine.

The invention will now be described in more detail with reference to thefollowing Examples which merely serve to illustrate the invention, notto restrict it.

EXAMPLE 1

0.2 g starch were dissolved in 1.0 ml formamide by heating to about 60°C. The clear solution was allowed to cool to room temperature, whereupon70 mg magnetite particles (Ferrofluid Corp., Nashua, N.H., USA) wereadmixed to the starch solution. The solution was transferred to a 100 mlbeaker containing 50 ml xylene/CHCl₃ (4:1) in which the emulsifiersPluronic F-68® and Pluronic L-35® had been dissolved, both of which hada concentration of 2.5% (weight volume). The mixture was emulsifiedultrasonically (Ultrasonic 350 G, 350 watt) for 1 min., whereupon theresulting emulsion was transferred to 400 ml acetone in which theemulsifier Tween 80® had been dissolved to a concentration of 0.1%(weight volume). Transfer of the emulsion to the acetone-Tween 80®solution was effected by pouring the emulsion in the form of a finestream into the acetone under stirring at a rate of about 1000 r/min.The crystallized magnetic nanospheres were washed 4 times more with thesaid acetone solution, whereafter they were dried under rotationalevaporation or freeze drying or, alternatively, were kept in the acetonesolution.

EXAMPLE 2

Example 1 was repeated, but with the difference that instead of 70 mgmagnetite particles 100 μl of water-based "Ferrofluid" were admixed tothe starch solution (Catalogue No. A-01, 400 gauss from FerrofluidCorp., Nashua, N.H., USA).

EXAMPLE 3

Example 1 was repeated, but with the difference that 140 mg magnetiteparticles were admixed to the starch solution instead of 70 mg.

EXAMPLE 4

Example 1 was repeated, but with the difference that maize oil alone wasused instead of xylene/CHCl₃ (4:1) with the emulsifiers Pluronic F-68®and Pluronic L-35®.

EXAMPLE 5

Example 1 was repeated, but with the difference that the starch wascrystallized in ethanol with the addition of 1 g Tween 80® per liter ofethanol, instead of acetone.

EXAMPLE 6

Example 1 was repeated, but with the difference that 0.45 g starch wasdissolved in 0.1 ml DMSO instead of 0.2 g in formamide.

EXAMPLE 7

Example 1 was repeated, but with the difference that 0.4 g dextran(molecular weight 40,000 from Pharmacia AB) was dissolved in 1.00 mlwater instead of 0.2 g starch in formamide.

EXAMPLE 8

Example 1 was repeated, but with the difference that 1.5 g lactose wasdissolved in 1 ml water, and that the lactose solution was emulsified inoil.

EXAMPLE 9

Example 1 was repeated, with the difference that 1.5 g maltose wasdissolved in 1 ml water, and that the maltose solution was emulsified inoil.

EXAMPLE 10

Example 1 was repeated, with the difference that 2.5 g glucose weredissolved in 1 ml water, and that the glucose solution was emulsified inoil.

EXAMPLE 11

The magnetic nanospheres obtained in any one of Examples 1-10 were mixedwith ³ H-vincristine, whereupon ³ H-vincristine released by diffusionfrom the nanospheres was measured in vitro in a 0.1M Na-phosphate bufferat pH 7.5. After an initially high diffusion from the nanosphere, arelatively uniform leakage was obtained, and after 4 hours 30% of theadded amount of ³ H-vincristine were still adsorbed to the nanospheres.

EXAMPLE 12

1 g of a 50% (weight volume) aqueous solution of dextran having amolecular weight of 40,000 was mixed with 70 mg magnetite particles and100 μl of an ovalbumin solution containing 100 mg ovalbumin/ml water. 5μl ¹²⁵ I-labelled ovalbumin had previously been added to the lattersolution.

The dextran-ovalbumin solution was suspended in 25 ml vegetable oil in a100 ml beaker and cooled to +4° C. The mixture was emulsifiedultrasonically for 1 min., whereupon the emulsion was poured into 200 mlacetone in which the emulsifier Tween 80® had been dissolved to aconcentration of 0.1% (weight volume). While the emulsion was beingcarefully poured into the acetone solution, the latter was stirred at arate of about 1000 r/min. The resulting dextran spheres stabilized bycrystallization and enclosing ovalbumin and magnetite were washed 4times more with the said acetone solution, whereupon they wereair-dried.

Normally, such an experiment gives a yield of about 250 mg spheres where60-70% of ovalbumin added are enclosed in the carbohydrate matrix.

EXAMPLE 13

Example 1 was repeated, but with the difference that interferon was usedinstead of ovalbumin.

EXAMPLE 14

Example 1 was repeated, but with the difference that plasmin was usedinstead of ovalbumin.

EXAMPLE 15

The nanospheres produced in Example 1 were used for covalently bondinghigh-molecular substances of the protein type. 1 ml nanospheres wasactivated with 200 μl epibromohydrin for 4 hours in 10 ml 0.1M NaOH.After washing, ¹²⁵ I-myoglobin was added and allowed to coupleovernight. Unbonded myoglobin was washed off with 1 mM HCl, 0.3M NaCland 0.1M NaHCO₃, pH 9.5, whereupon the activity was determined. 0.7 mgmyoglobin had been covalently coupled to the starch matrix.

Under the action of alpha-amylase in a concentration which was 100 timeshigher than in normal human serum, about 30% of coupled ¹²⁵ I-myoglobinhad been released from the matrix after 24 hours at room temperature andin 0.1M Na-phosphate buffer, pH 7.5.

In vivo experiment

The magnetic nanospheres obtained in Example 1 were used for in vivotesting on rats.

To test the half-life in the circulation, the rat was anaesthetised with5% chloral injected intraperitoneally, whereupon v. femoralis and thecentral tail artery were both catheterised cranially. The radioactivelylabelled magnetic nanospheres were injected via v. femoralis, and bloodsamples were taken via the tail artery at predetermined time intervals,whereupon the circulating amount of magnetic nanospheres was determined.The half-life obtained showed that the majority of the number ofmagnetic nanospheres had such a long half-life in the circulation thatthey can be concentrated in an organ of the body to an extent sufficientto attain therapeutical concentration of the transported,pharmaceutically active preparation.

In another experiment, the rat was killed after a predetermined time,whereupon the amount of magnetic nanospheres in different organs, interalia the brain, was determined. This experiment was repeated, but thistime with the rat's head positioned in a magnetic field of 1 Tesla, anda marked increase of the radioactivity in the brain was observed.

We claim:
 1. A nanosphere or nanoparticle for intravascularadministration, which is magnetically responsive and biologicallydegradable and which is made up of a matrix in which a magnetic materialis enclosed, characterized in that said nanosphere or nanoparticle hasan average diameter which does not exceed 1500 nm, and circulates in thevascular system after administration thereto, said matrix comprising ahydrophillic, crystalline carbohydrate.
 2. A nanosphere or ananoparticle as claimed in claim 1, characterised in that the magneticmaterial is present in the form of particles or a colloid.
 3. Ananosphere or a nanoparticle as claimed in claim 2, characterised inthat the magnetic material consists of magnetite particles having anaverage diameter of 1-1000 nm, preferably 10-20 nm.
 4. A nanosphere or ananoparticle as claimed in one or more of claims 1, 2, or 3,characterised in that the crystalline carbohydrate is glucose, maltoseor lactose.
 5. A nanosphere or a nanoparticle as claimed in one or moreof claims 1, 2, or 3, characterised in that the crystalline carbohydrateis a carbohydrate polymer, preferably with alpha(1-4) bonds between thecarbohydrates comprised by the polymer.
 6. A nanosphere or ananoparticle as claimed in claim 5, characterised in that thecarbohydrate polymer is starch, glycogen, pullullan or a derivativethereof.
 7. A nanosphere or a nanoparticle as claimed in claim 5,characterised in that the carbohydrate polymer is dextran or aderivative thereof.
 8. Use of the nanosphere or the nanoparticle asclaimed in claim 1 for transport and concentration of pharmacologicallyactive substances in biological systems, preferably animal systems.